The present invention, in some embodiments thereof, relates to methods of identifying trophoblasts in a biological sample such as a maternal blood and, more particularly, but not exclusively, to utilizing same for prenatal diagnosis.
Prenatal diagnosis involves the identification of chromosomal abnormalities and/or genetic diseases in a human fetus. The current practice for detecting chromosomal aberrations such as presence of extra chromosomes [e.g., the most common condition, Trisomy 21 (Down's syndrome); Klinefelter's syndrome (47, XXY); Trisomy 13 (Patau syndrome); Trisomy 18 (Edwards syndrome); 47, XYY; 47, XXX], absence of chromosomes [e.g., Turner's syndrome (45, X)], various translocations and deletions, as well as diagnosis of genetic diseases (e.g., Cystic Fibrosis, Tay-Sachs Disease) involves invasive procedures, mainly chorionic villus sampling (CVS) and/or amniocentesis.
Because of the increased risk of Down's syndrome in pregnancies of advanced maternal age, in most Western World countries, prenatal diagnosis is offered to women over the age of 35 as well as to women who are known carriers of genetic diseases or chromosomal aberrations (e.g., balanced translocations). Thus, the risk of a pregnant female older than 35 years old to give birth to an infant with a detectable chromosomal aberration has been drastically reduced. However, due to the lack of prenatal diagnosis in younger women, about 80% of Down syndrome infants are born to pregnant females under the age of 35.
CVS or amniocentesis are invasive procedures which carry 2-4% or 0.5-1.0%, respectively, of procedure-related risks of miscarriage. In these procedures the sampled fetal cells are either directly tested by FISH/DNA analyses, or expanded in culture and then subjected to karyotype analyses (e.g., by G-banding).
In attempts to develop a less invasive prenatal diagnosis, various studies have identified markers which can detect fetal cells in the cervical canal (transcervical cells).
Transcervical cells can be retrieved using aspiration, cytobrush, cotton wool swabs, endocervical or intrauterine lavage and be subject to analysis using trophoblast-specific antibodies such as FT1.41.1, PLAP, NDOG-1, NDOG-5, mAb 340 and the human leukocyte antigen (HLA-G) antibody.
PCT Publication No. WO2006/018849 discloses methods of prenatal diagnosis performed on transcervical cells using the HLA-G, PLAP, CHL1 and/or NDOG-1 antibodies followed by FISH analyses.
Non-invasive prenatal diagnosis using the maternal blood has been also attempted. Although rare (e.g., one fetal cell per million nucleated maternal blood cells), fetal trophoblasts, leukocytes and nucleated erythrocytes were found in the maternal blood during the first trimester of pregnancy. However, the isolation of trophoblasts and leukocytes from the maternal blood is limited by the availability of fetal-specific antibodies. In addition, studies have shown that at least 50% of the nucleated red blood cells (NRBCs) isolated from the maternal blood are of maternal origin and moreover, certain cell types tend to persist in the maternal circulation and therefore potentially interfere with diagnosis of subsequent pregnancies (Bianchi D 1996, Troeger C, et al., 1999; Guetta et al., 2004).
Trophoblasts present an attractive target cell type for non-invasive prenatal diagnosis since they can be isolated from a maternal blood early in the first trimester, are distinguishable from maternal blood cells due to their unique structure and are absent in normal adult blood.
Trophoblasts have been isolated from maternal blood by several different methods related to surface antigen expression (e.g., HLA-G) and cell size (van Wijk et al., 2001; Vona et al., 2002). In addition, the present inventors have demonstrated the potential of trophoblasts isolated from the maternal blood to proliferate in vitro under suitable conditions (Guetta, et al., 2005). However, since HLA-G is not exclusively expressed in trophoblasts (Kirszenbaum M et. al. 1995) its use for detection of trophpoblasts in the maternal blood is limited.
Cytokeratins 7 and 8 (CK-7 and CK-8) were found to be expressed in placental tissues and cells (Maldonado-Estrada, J., et al., 2004; Yamamoto R, et al., 1990; Tamai Y., et al., 2000). In addition, the expression level of cytokeratin-19 (CK-19) in the serum throughout the third trimester was associated with severe pre-eclampsia (Tempfer C B., et al., 2000).
Additional background art includes Masuda J., et al., 2004; Daya, D. and Sabet, L. 1991; Oudejans C B, et al., 2003.